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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A theory is presented to describe the coupling of the directional movement of ATPase-containing translocases (such as helicases) along polymeric lattices to the steady-state kinetic parameters of the ATPase activity that drives this movement. This theory was developed to explain the results of an experimental investigation of one such enzyme, the bacteriophage T4 gene 41 protein helicase. The salient feature of the theory is that it correctly predicts the dependence of the rate of ATP hydrolysis by ATP-driven translocases on the length of polymer lattices along which they move. In the steady-state rate equation: [formula: see text] either Vmax, or K(act), or both, may depend on the lattice length. Two translocation models are considered. The first is a simple mechanism of the type E<-->E-Lat-->E, where the E-Lat-->E step represents the sum of the translocation steps of the enzyme along, and enzyme release from, the lattice. In the second model this mechanism is expanded to add an additional kinetic step, either before or after the translocation process. Variants of this second model can be used to represent the most simple translocase mechanisms. Another method of measuring the lattice length dependence of an ATP-driven translocase, which is applicable particularly to ATPases moving along DNA lattices, involves the use of lattice-binding proteins (such as single-stranded DNA binding proteins) that can block the movement of the translocases and therefore simulate lattice ends. In this protocol the dependence of the ATPase kinetics of the translocase on lattice length can be studied by experiments on long lattices complexed with lattice-binding proteins to various binding densities. This method is not always as unambiguous as direct measurement of ATPase activity on lattices of defined length, but can help to discriminate between mechanisms. The significance of the steady-state kinetic parameters obtained in such experiments is discussed in terms of the mechanistic rate constants that define the models we have investigated.
J Mol Biol 1994 Feb 04
PMID:Kinetic theory of ATP-driven translocases on one-dimensional polymer lattices. 810 84

The bacteriophage T4 gene 41 helicase protein (gp41) carries a single-stranded DNA-dependent ATPase activity that is essential to its helicase activity. This ATPase activity can be stimulated by a wide variety of single-stranded DNA cofactors, including homo-oligomers and homopolymers 8 to approximately 10,000 nucleotide residues in length, and by natural single-stranded DNA, such as bacteriophage M13 DNA. The steady-state ATPase activity of gp41 on single-stranded homopolymeric cofactors is dependent on the length of the cofactor, in that the kinetic parameters Vmax and K(act) (or KmDNA) have a characteristic length dependence. Vmax values for different DNA lengths show a hyperbolic dependence on DNA length, while K(act) values are independent of DNA lengths exceeding approximately 20 nucleotide residues. Use of the detailed theoretical analysis developed in the preceding paper reveals that: (1) these results support the earlier proposal that gp41 translocates on single-stranded DNA in an ATP-dependent manner; (2) translocation is undirectional; (3) translocation is processive to an extent that depends on the base composition of the DNA employed, with the average distance translocated per binding event ranging from 60 to 700 nucleotide residues; and (4) the detailed translocation mechanism of gp41 includes an obligatory slow step before or after the ATP-driven translocation process. Defined lengths of natural and homopolymer single-stranded DNA have also been created as gaps of known length distribution between clusters of gene 32 protein (gp32) bound along long single-stranded DNA molecules. ATPase data obtained with cofactors of this type also show unidirectional ATP-driven translocation of gp41 on both natural and homopolymeric single-stranded DNA. Direct binding studies of gp41 to short dT oligomers reveal two further features of the interaction of gp41 to single-stranded DNA: (1) nucleoside triphosphate binding is necessary for the formation of stable gp41-ssDNA complexes; and (2) the DNA binding site size of gp41 is between 12 and 20 nucleotide residues per protein monomer. Possible translocation mechanisms for gp41 are discussed within the context of these results.
J Mol Biol 1994 Feb 04
PMID:Kinetic parameters of the translocation of bacteriophage T4 gene 41 protein helicase on single-stranded DNA. 810 85

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.
Mol Cell Biol 1994 Apr
PMID:The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence. 813 36

The human ERCC3 gene, which corrects specifically the nucleotide excision repair defect in human xeroderma pigmentosum group B and cross-complements the repair deficiency in rodent UV-sensitive mutants of group 3, encodes a presumed DNA helicase that is identical to the p89 subunit of the general transcription factor TFIIH/BTF2. To examine the significance of the postulated functional domains in ERCC3, we have introduced mutations in the ERCC3 cDNA by means of site-specific mutagenesis and have determined the repair capacity of each mutant to complement the UV-sensitive phenotype of rodent group 3 cells. A conservative substitution of arginine for the invariant lysine residue in the ATPase motif (helicase domain I), six deletion mutations in the other helicase domains, and a deletion in the potential helix-turn-helix DNA-binding motif fail to complement the ERCC3 excision repair defect of rodent group 3 mutants, which implies that the helicase domains as well as the potential DNA-binding motif are required for the repair function of ERCC3. Analysis of carboxy-terminal deletions suggests that the carboxy-terminal exon may comprise a distinct determinant for the DNA repair function. In addition, we show that a functional epitope-tagged version of ERCC3 accumulates in the nucleus. Deletion of the putative nuclear location signal impairs neither the nuclear location nor the repair function, indicating that other sequences may (also) be involved in translocation of ERCC3 to the nucleus.
Mol Cell Biol 1994 Jun
PMID:Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. 819 50

The Staphylococcus aureus chromosomal gene pcrA, identified by mutations, such as pcrA3, that affect plasmid pT181 replication, has been cloned and sequenced. The pcrA gene encodes a protein with significant similarity (40% identity) to two Escherichia coli helicases: the helicase II encoded by the uvrD gene and the Rep helicase. The pcrA3 mutation was found to be a C to T transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. The pcrA gene seems to belong to an operon containing at least one other gene, tentatively named pcrB, upstream from pcrA. The PcrA protein was shown to be essential for cell viability and overproduction has deleterious effects on the host and plasmid replication.
Mol Gen Genet 1993 Oct
PMID:Characterization of the Staphylococcus aureus chromosomal gene pcrA, identified by mutations affecting plasmid pT181 replication. 823 3

DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.
Mol Pharmacol 1993 Nov
PMID:Antihelicase action of DNA-binding anticancer agents: relationship to guanosine-cytidine intercalator binding. 824 9

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Plant Microbe Interact
PMID:Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene. 827 74

A site-specific lysine to methionine mutation has been engineered at the invariant Lys35 residue in the ATPase A binding site of the Escherichia coli uvrD gene encoding DNA helicase II. The mutant protein (UvrDK35M) has been purified to apparent homogeneity and characterized. The kcat for DNA-dependent ATP hydrolysis was less than 0.5% that of the wild-type enzyme with no change in the apparent Km for ATP. No unwinding of partial duplex DNA substrates could be detected using the mutant protein. Moreover, the mutant protein inhibited the unwinding reaction catalyzed by the wild-type protein at ratios of mutant enzyme to wild-type enzyme < 1. We conclude that the K35M mutation renders helicase II catalytically inactive as a DNA helicase with little or no effect on the ability of the enzyme to bind ATP, DNA, or other proteins. In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway. Additional genetic characterization of the uvrDK35M allele, supplied on a plasmid, suggests that expression of the mutant protein, at levels equivalent to that of the wild-type protein, results in a dominant negative phenotype. Expression of lower levels of the mutant protein, both in the presence and absence of wild-type helicase II, results in a constitutive induction of the cellular SOS response and extensive filamentation of cells. This induction of the SOS response is not due to a defect in methyl-directed mismatch repair. Taken together, these data are consistent with the notion that E. coli helicase II may have a role in DNA replication.
J Mol Biol 1994 Jan 14
PMID:A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization. 828 72

The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOBw) has been analyzed by Tn5tac1 insertion mutagenesis, genetic complementation and DNA sequencing. Three genes (trwA, trwB and trwC) were mapped within MOBw. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13.5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases. It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.
J Mol Biol 1994 Jan 14
PMID:Genetic organization of the conjugal DNA processing region of the IncW plasmid R388. 828 74

Protein kinase activity was revealed in complex forms of rat liver DNA polymerase alpha containing 3'-5'-exonuclease, primase, helicase, DNA ligase. Protein kinase (mol. mass about 200 kDa) has been partially purified from a specimen of high molecular mass DNA polymerase alpha of nuclear membrane of regenerating liver. The protein kinase activity of the complex form of DNA polymerase alpha was maximal in the cytosol in normal rat liver cells and in the nuclear membrane in dividing cells (40 h after partial hepatectomy). The main phosphokinase properties of this enzyme were determined.
Mol Biol (Mosk)
PMID:[Isolation of protein phosphokinase from a complex form of DNA polymerase alpha from rat liver]. 831 39


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